The present invention relates to a method for covalent attachment of antibodies or other molecules to a solid support using extended length heterobifunctional reagents.
In diagnostic assays, the reaction between a specific binding member and its complement is often employed to detect whether (and in some assays, how much) specific binding member or complement is present in a sample. In one type of diagnostic assay, a specific binding member (e.g. an antibody) is detected in a sample by introducing its complement (e.g. an antigen) into the sample and determining if any reaction occurs between the two reagents. Alternatively, the complement itself can be detected in a sample by introducing the specific binding member into the sample and determining whether any reaction occurs. Because it is often difficult to detect whether any reaction has occurred, a second specific binding member may be added to the sample. The second specific binding member can react either with the first specific binding member or its complement, and the second member bears a detectable label. Of course, it is impossible to determine beforehand how much labelled second specific binding member must be added because it is unknown how much, if any, of the substance to be detected is in the sample. Thus, the labelled specific binding member is added in excess of the maximum concentration of the substance typically found in such samples. However, the labelled specific binding member which does not bind with the substance must be separated from the sample so that only the bound labelled member is detected, indicating that the substance is indeed in the sample.
A common approach to separate bound from unbound labelled member is to employ solid phase separation. A typical example of such separation involves linking the first specific binding member (in the case of assays for complement to the first member) or complement (in the case of assays for specific binding member) to a solid phase (such as microparticles) which can be separated from the sample, for example, by filtration or gravity sedimentation. The label associated either with the solid phase or still in the sample is proportional to the amount of substance to be detected in the original sample.
Other variations to this general solid phase separation scheme have been developed, but most such schemes involve the binding of the substance to be analyzed to a specific binding member linked to a solid phase. This binding is crucial to assay performance. However, the linkage between the solid phase and the specific binding member can subsequently affect binding of the substance to be analyzed. An example will illustrate the point. Antibodies have extremely specific structural, spacial, and polar configurations which endow them with the ability to recognize and bind to one type of analyte, and virtually none other. When antibodies are employed in assays for the detection of antigens, antibodies can be linked to solid phases. However, the proximity of the solid phase to the antibody can block sites on the antibody where antigen binds. Alternatively, the linkage (usually covalent) between the antibody and solid phase can alter the structure (conformation) of the antibody so that the linkage may deleteriously affect binding of the antibody to the analyte. The same situation holds for conjugation of analytes, particularly proteins, to solid phases. The analyte conformation can change upon conjugation so the free antibody in the sample can no longer recognize it.
Covalent attachment of proteins to solid phases using heterobifunctional reagents has been accomplished with mixed results in the past. In some cases the proteins were directly conjugated to the solid phase. Generally, the connecting tether has been quite short in comparison with the size of the bound protein. This is disadvantageous in that the bound protein can still be hindered in performing its biological function due to steric crowding, inaccessability of binding sites, etc. This has been a problem which has limited the bioactivity and stability of derivatized solid phases in the past.